Wisconsin Alumni Research Foundation

药物发现 & 发展
Research Tool for Protein Conformation Analysis
WARF: P160180US02

十大正规靠谱网赌平台者:迈克尔•苏斯曼, 犹大Shohet, " Choudhury, 约书亚Blatz, 本杰明明柯夫, 丹尼尔·本杰明

The Wisconsin Alumni Research Foundation (WARF) is working with UW–Madison researchers to develop a powerful new method that combines plasma-induced oxidation, 然后是质谱分析, to study the 3-D conformation and solvent accessibility of biological molecules.
The three-dimensional structure of proteins can be studied in solution using various methods of covalent and noncovalent labeling in which the readout is accomplished by mass spectrometry (MS). 这些程序, collectively known as protein ‘footprinting,’ all involve measuring the solvent accessibility of either the peptide backbone (hydrogen deuterium exchange) or amino acid side chains (covalent modification and cross linking reagents). 标签后, proteins are analyzed using tandem MS and linkages or adducts are quantitatively mapped to specific regions, revealing areas and degrees of intermolecular interactions or solvent accessibility.

The emergence of the quickly expanding protein therapeutics industry has generated a need for easy, sensitive and reproducible methods for detecting changes in protein conformation as part of the rigorous 质量 control process required for clinical use and for quickly detecting the regions of proteins involved in their binding to ligands and/or protein-protein interactions.
UW–Madison researchers have developed a method and easy-to-operate device that uses plasma to perform hydroxyl radical footprinting. The device tags the outer surface of the protein and allows the user to study its 3-D conformation via mass spectrometry.

这项新技术, 哪一种方法可以在台式电脑上使用, applicable to a range of protein concentrations and sizes and generates µs bursts of hydroxyl radicals without added 化学物质 or reagents, has been developed and the results benchmarked. It is useful for quickly performing epitope mapping or assessing protein structural characteristics such as unfolding and conformational changes. The method can be used with two or more distinct proteins to map binding events, which enables pharmaceutical and R&D labs to image proteins in their natural state.

The researchers believe this tool will enable much quicker turnaround (on the order of hours) than X-ray crystallography and more reliable data than Hydrogen-Deuterium Exchange (HDX). It can be manufactured alone or in conjunction with mass spectrometry systems.
  • Assessing the 3-D conformation of proteins, nucleic acids and other biological molecules
  • Determining solvent accessibility of different parts of the protein
  • Study of condition-dependent sample properties (e.g.动力学,蛋白质折叠)
  • Demonstrated dose-dependent and reproducible results
  • 独特的设备设计和方法
  • Shown to work quickly and efficiently
  • Simple to operate and relatively inexpensive to produce
  • No exogenous 材料 need to be added to the sample solution.
    • Users can test various conditions of interest without artifacts.
这项新技术 was first benchmarked with model compounds, and then applied to a biological problem, i.e., ligand (EGF) induced changes in the conformation of the external (ecto) domain of Epidermal Growth Factor Receptor (EGFR). Regions in which oxidation decreased upon adding EGF fall along the dimerization interface, consistent with models derived from crystal structures. These results demonstrate that plasma-generated hydroxyl radicals from water can be used to map protein conformational changes, and provide a readily accessible means of studying protein structure in solution.

Future experiments will explore optimization and further biological applications. The researchers envision optional modifications to the apparatus to allow adjustments to the frequency, 脉冲宽度和脉冲数, 脉冲的电压和时间. Such adjustments would enhance the flexibility of this powerful analytical technology.
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